Monoclonal antibodies often achieve their therapeutic benefit through two binding events. First, the variable domain of the antibody binds a specific protein on a target cell. This is followed by recruitment of effector cells that bind to the constant region (Fc) of the antibody and destroy cells to which the antibody is bound.
The potency of an antibody (or other immunotherapeutic composition) depends on multiple mechanisms of action, including those mediated by effector cells expressing Fc receptors (FcRs). Fc receptors have activating or inhibitory functional roles and differ in their distribution among effector cells. Monocytes, macrophages, and neutrophils express both activating and inhibitory FcRs, whereas natural killer (NK) cells solely express the activating FcRIIIa. Thus, the degree to which an antibody (or other immunotherapeutic) can engage the various Fc receptors are important for clinical outcome.
Amino acid- and glyco-engineering of the antibody Fc domain are two ways to modify effector cell functions of antibodies and other immunothereapuetics. See, e.g., Chu et al., Molecular Immunology 45:3926-3933 (2008).
It would be desirable to engineer an antibody or Fc fusion protein comprising improved properties. For example, it would be desirable to engineer antibodies or other immunotherapeutics which bind to Fc gamma receptor IIB (CD32B), but which do not bind (or binds with reduced affinity) to Fc gamma receptor IIA (CD32A) and Fc gamma receptor IIIA (CD16A) and Fc gamma receptor I (CD64). Such antibodies would be characterized by their lack of (or a significant decrease in) effector function and increased anti-inflammatory properties.
The presence of N-glycosylation not only plays a role in the effector function of an antibody, the particular composition of the N-linked oligosaccharide is also important for its end function. For example, the lack of fucose or the presence of bisecting N-acetyl glucosamine has been positively correlated with the potency of the ADCC, Rothman (1989), Umana et al., Nat. Biotech. 17: 176-180 (1999), Shields et al., J. Biol. Chem. 277: 26733-26740 (2002), and Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003). There is also evidence that sialylation in the Fc region is positively correlated with the anti-inflammatory properties of intravenous immunoglobulin (IVIG). See, e.g., Kaneko et al., Science, 313: 670-673, 2006; Nimmerjahn and Ravetch, J. Exp. Med., 204: 11-15, 2007.
Given the utility of specific N-glycosylation in the function and potency of antibodies, methods for modifying the composition of N-linked oligosaccharides of antibodies and method of modifying the effector functions of antibodies and other immunotherapeutics would be desirable.
Yeast and other fungal hosts are important production platforms for the generation of recombinant proteins. Yeasts are eukaryotes and, therefore, share common evolutionary processes with higher eukaryotes, including many of the post-translational modifications that occur in the secretory pathway. Recent advances in glycoengineering have resulted in cell lines of the yeast strain Pichia pastoris with genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the process of glycosylation in humans. See, for example, U.S. Pat. Nos. 7,029,872, 7,326,681 and 7,449,308 that describe methods for producing a recombinant glycoprotein in a lower eukaryote host cell that are substantially identical to their human counterparts. Human-like sialylated bi-antennary complex N-linked glycans like those produced in Pichia pastoris from the aforesaid methods have demonstrated utility for the production of therapeutic glycoproteins. Thus, a method for further modifying or improving the production of antibodies in yeasts such as Pichia pastoris would be desirable.